Even in a highly specific immunological measurement method utilizing an antigen-antibody reaction, there has been a problem that some samples which do not contain an antigen to be measured show positive measured values, i.e., measured values different from the true values. This phenomenon is called a non-specific reaction.
As an immunological measurement method utilizing an insoluble carrier on which an antibody specifically binding with an antigen to be measured is immobilized, a latex agglutination optical measurement method and an enzyme immunoassay are known. When such a method is used to measure the concentration of an antigen contained in samples, there are some cases where certain samples contain a factor which is different from the antigen, but can recognize and react with the immobilized antibody (a non-specific reaction factor). In these cases, such samples to be measured show positive measured values, i.e., measured values different from the true values.
The non-specific reaction factor contained in samples is not particularly limited, so long as it is a substance which is different from an antigen to be measured, and can react with an antibody-immobilized carrier. Examples of the non-specific reaction factor which frequently occurs include naturally occurring antibodies such as IgM, IgG, and IgA. When a sample is a human body fluid, such as a serum or plasma, human IgM or human IgG frequently participates in non-specific reactions, and a non-specific agglutination of latex carriers occurs in the latex agglutination optical measurement method.
As a method of inhibiting a non-specific reaction caused by the non-specific reaction factor, a method of avoiding the effect of human IgM or human IgG by supplementing a measurement reagent with an anti-human-IgM antibody, an anti-human-IgG antibody, or the like. As a concrete inhibitor added in this method, a serum component obtained from animals other than humans is proposed (patent reference 1). Patent reference 2 discloses a method of supplementing a measurement reagent with an antibody obtained by immunizing an animal with a non-specific reaction factor. A non-specific reaction can be decreased by supplementing a measurement reagent with this type of antibody.
However, IgG or IgM obtained from an animal serum has multiple sites for the recognition of an antigen. For example, a molecule of IgG has two antigen recognition sites, and a molecule of IgM has at least ten antigen recognition sites. In addition, IgG and IgM exhibit a highly hydrophobic property, in comparison with other proteins. For these reasons, when IgG or IgM coexists in the same reaction liquid with an antigen which is a target of IgG or IgM, an immunological nephelometric reaction occurs. The immunological nephelometric reaction is a phenomenon that multiple antigens are crosslinked with IgG or IgM to form a huge immunological complex, which causes cloudiness capable of being optically detected as turbidity. For example, when human IgM is added to a reaction liquid containing an antibody specific to human IgM, an immunological nephelometric reaction occurs and the reaction liquid becomes cloudy. Under these conditions, when a latex agglutination optical measurement method in which the amount of an antigen is determined by optically measuring turbidity is carried out, sometimes an accurate measured value cannot be obtained due to the immunological nephelometric reaction. As described above, a non-specific reaction due to a non-specific reaction factor can be avoided, but there remains a problem that the avoidance newly results in a secondary immunological nephelometric reaction. In addition, there is another problem that when a sample contains rheumatoid factors, the immunological nephelometric reaction is increased because the rheumatoid factors bind with the Fc region of an IgG or IgM molecule.
These problems can be solved by adding a decreased amount of antibody to a measurement reagent. However, when the amount added is less than the amount sufficient to inhibit the effect of a non-specific reaction factor, the effect of inhibiting the non-specific reaction is insufficient.
Under these circumstances, the present inventors examined a method utilizing, as a non-specific reaction inhibitor, an “antibody fragment” obtained by digesting an “antibody”, for example, F(ab′)2 obtained by digesting an IgG molecule with a protease, pepsin. An IgG or IgM molecule contains an Fc region having a high hydrophobicity, but F(ab′)2 does not contain the Fc region. Therefore, when F(ab′)2 is used, the immunological nephelometric reaction caused by the addition of an antibody does not easily occur, and therefore, a large amount of F(ab′)2 can be added to a measurement reagent. In addition, the effect of rheumatoid factors can be avoided because F(ab′)2 does not contain the Fc region. Therefore, the above problems caused by the addition of IgG (i.e., the immunological nephelometric reaction and the effect of rheumatoid factors) can be avoided by utilizing F(ab′)2. Under these circumstances, the effect of F(ab′)2 of inhibiting a non-specific reaction was the same as that of IgG. As described above, the method utilizing an antibody fragment F(ab′)2 as a non-specific reaction inhibitor is more practical than the invention utilizing an antibody. The present inventors further evaluated a measurement reagent containing the F(ab′)2 as the inhibitor, it was found that the measurement reagent has a disadvantage in maintaining the effect of inhibiting a non-specific reaction.
The F(ab′)2 molecule is a molecule in which two molecules of Fab′ are linked via a disulfide bond of the hinge region. F(ab′)2 is characterized by a high sensitivity to an oxidation-reduction reaction. F(ab′)2 is easily reduced and degraded into two molecules of Fab′. Further, because a serum component contains a protease which cleaves a peptide bond at the hinge region of F(ab′)2, F(ab′)2 is degraded if the purification of F(ab′)2 from a serum is insufficient or if the measurement reagent is contaminated with the protease or the like. Therefore, when a measurement reagent coexists with F(ab′)2, F(ab′)2 is easily degraded in accordance with a method of storing the measurement reagent. Because Fab′ exhibits a very weak effect of inhibiting a non-specific reaction in comparison with an antibody or F(ab′)2, it was considered that the degradation of F(ab′)2 in the measurement reagent reduced the maintenance of the effect of inhibiting a non-specific reaction. Actually, the effect of inhibiting a non-specific reaction was not significantly observed in a measurement reagent supplemented with Fab′ as a non-specific reaction inhibitor.
As prior art different from the above-mentioned inhibition of a non-specific reaction in an immunological measurement, a use of chemically-modified Fab′ for an antitumor drug is known. For example, Delgado C. et al. (non-patent reference 1) disclose an antitumor drug containing Fab′ chemically modified with polyethylene glycol. Patent reference 3 discloses an antitumor drug containing Fab′ linked with a drug and a polymer via thiol groups of Fab′.    [patent reference 1] Japanese Unexamined Patent Publication (Kokai) No. 2006-38823    [patent reference 2] Japanese Unexamined Patent Publication (Kokai) No. 11-287801    [patent reference 3] U.S. Pat. No. 5,541,297    [non-patent reference 1] British Journal of Cancer, (United Kingdom), 1996, vol. 73, no. 2, p. 175-182